CN112029677B - GKRZ vibrio capable of highly producing biological nano-magnetic particles and application thereof - Google Patents
GKRZ vibrio capable of highly producing biological nano-magnetic particles and application thereof Download PDFInfo
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- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
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Abstract
GKRZ vibrio with high yield of biological nano-magnetic particles, wherein the preservation number of the GKRZ vibrio with high yield of biological nano-magnetic particles is CGMCC No. 19304; the GKRZ vibrio can be cultured in large-scale industrial fermentation, the growth period is short, specifically 48 hours, biological nano-magnetic particles are produced with high yield, the biological nano-magnetic particles produced by each 1g of bacteria are 25-30mg, and the produced biological nano-magnetic particles can be used for in vivo and in vitro diagnostic reagents, drug carriers and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a GKRZ vibrio with high yield of biological nano-magnetic particles and application thereof.
Background
The biological nano magnetic particles generally have super-strong paramagnetism, can be rapidly gathered in a magnetic field, have uniform particle size, ensure strong enough magnetic responsiveness and can not settle, have rich surface active groups, can be coupled with biochemical substances, and are widely used in a plurality of fields of immunoassay, nucleic acid separation and extraction, cell sorting, enzyme fixation and the like.
At present, most of biological nano magnetic particles are derived from magnetotactic bacteria, but most of the magnetotactic bacteria grow slowly, the living environment simulation is difficult, the culture difficulty is high, the production period is long, and large-scale culture is difficult.
Disclosure of Invention
The invention aims to overcome the problems of slow growth, long culture time and difficulty in large-scale culture of biological nano-magnetic particles, screen and culture a novel GKRZ vibrio of the biological nano-magnetic particles, and provide a corresponding extraction method of the biological nano-magnetic particles and research on the aspect of drug loading of the biological nano-magnetic particles.
The specific technical scheme of the invention is as follows:
the invention provides a GKRZ vibrio with high-yield biological nano-magnetic particles, the preservation number of the GKRZ vibrio with the high-yield biological nano-magnetic particles is CGMCC No.19304, the strain is preserved in China general microbiological culture Collection center in 1 month and 3 months in 2020, and the addresses are as follows: beijing, Chaoyang, Beichen Xilu No.1 Hospital No. 3, the strain name is GKRZ, and Latin's name is Vibrio.
The strain is negative in gram stain, anaerobic, acidophilic, rod-shaped, has the size of (0.4-0.6 mu m) x (1-3 mu m), can grow in a liquid culture medium and a solid culture medium by taking silicate as a main energy substance, and has black bacterial liquid and milky white and smooth bacterial colony.
The culture medium of the technical scheme of the invention comprises the following components:
the invention also provides a GKRZ vibrio liquid culture medium for high-yield biological nano-magnetic particles, which comprises the following components:
each liter contains 3g of sodium pyruvate and 0.1g of KH 2 PO 4 0.15g of MgSO 2 4 .7H 2 O, 0.34g of NaNO 3 2.38g of hydroxyethyl piperazine ethanethiosulfonic acid, 3g of bean cake powder and 0.1g of yeast powder, and adjusting the pH value to 6.0-7.0; the incubation temperature was 28 ℃.
The strain provided by the invention is separated from a water sample of a Jingmi water canal and is obtained by screening and passage.
The strain provided by the invention is GKRZ vibrio separated from a water sample of a Jingmi aqueduct to obtain GKRZ vibrio, the GKRZ vibrio is inoculated in a liquid culture medium by the inoculation amount of 5-10% (v/v), is subjected to shake cultivation for 18h at 120rpm under the condition of 28 ℃ to logarithmic phase for later use, and is screened by using a liquid magnetophoresis separation method to obtain the GKRZ vibrio with high-yield biological nano-magnetic particles.
The application of the GKRZ vibrio with high yield of biological nano-magnetic particles in the production of recombinant biological nano-magnetic particle crystals.
The GKRZ vibrio with high yield of biological nano-magnetic particles is applied to drug loading.
The GKRZ vibrio with high yield of biological nano-magnetic particles is applied to the preparation of in vivo and in vitro diagnostic reagents or kits.
The culture method of the GKRZ vibrio with high yield of the biological nano-magnetic particles is to inoculate the GKRZ vibrio into a liquid culture medium for fermentation culture.
Further, the method for inoculating the Vibrio GKRZ into the liquid culture medium for fermentation culture comprises the following steps: vibrio GKRZ was inoculated into a liquid medium in an inoculation amount of (5)% - (10)% (v/v), and subjected to shaking culture at (120) rpm at (28) ° C to logarithmic phase.
The invention also provides a method for producing biological nano-magnetic particles, which comprises the following steps: (1) culturing the vibrio GKRZ of claim 1 or 2; (2) centrifugally collecting thalli; (3) adding buffer solution to homogenize and crush thallus and collecting biological nanometer magnetic particles; (4) adding buffer solution for ultrasonic cleaning, and performing magnetic separation to obtain purified biological nano magnetic particles.
Further, the centrifugation speed of the step (2) is 5000rpm, the time is 10min, the buffer solution of the step (3) is PBS, the mass ratio of the adding amount of the buffer solution to the GKRZ vibrio is 10:1, the buffer solution of the step (4) is PBS, and the ultrasonic cleaning condition is 40KHz and 30 min.
The invention also provides a method for preparing the biological nano magnetic particle drug carrier, which comprises the following steps: s1, culturing the Vibrio GKRZ; s2 preparing biological nano magnetic particles by using GKRZ vibrio; s3 coupling the biological nano-magnetic particle with the medicine.
Further, the drug is tuzumab, and the specific operation of step S3 is: ultrasonically coupling biological nano-magnetic particles with the SPDP in a mass ratio of 1:1 for 1min every time, repeating the ultrasonic coupling for 30 times, and cleaning for three times by using PBS with the pH value of 7.4; adding recombinant SPA with the same amount as the biological nano-magnetic particles and the SPDP, performing ultrasonic coupling for 1min each time, repeating for 30 times, and washing with PBS with pH of 7.4 for three times; and then adding trastuzumab with the same quantity as the biological nano magnetic particles and the SPDP for coupling, and placing in a shaking table at 37 ℃ for mixing for 2h to obtain the magnetic nanoparticle.
Wherein SPDP is 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester.
The invention also provides a method for screening the GKRZ vibrio capable of producing the biological nano-magnetic particles at high yield, which is to screen the GKRZ vibrio capable of producing the biological nano-magnetic particles at high yield by using a liquid magnetophoresis separation method.
Specifically, Race-track liquid magnetophoretic separation can be used, the separation time is 20min,
the GKRZ vibrio capable of producing the biological nano-magnetic particles with high yield can be subjected to large-scale industrial fermentation culture, the growth period is short, specifically 48 hours, the biological nano-magnetic particles with high yield are produced in an amount of 25-30mg per 1g of bacteria, and the produced biological nano-magnetic particles can be used for in vivo and in vitro diagnostic reagents, drug carriers and the like.
Drawings
FIG. 1 shows a photomicrograph of Vibrio GKRZ (magnification 30000X) with highly productive biological nano-magnetic particles;
FIG. 2 shows the microscope photograph of Vibrio GKRZ (80000X magnification) of high-yield biological nano-magnetic particles.
Detailed Description
Example 1
The embodiment provides GKRZ vibrio capable of producing biological nano-magnetic particles at high yield and application thereof, wherein the GKRZ vibrio is obtained by screening through a liquid magnetophoresis separation method, can be subjected to large-scale industrial fermentation culture, and has the advantages of short growth cycle, easiness in culture and the like, the growth cycle is 48 hours, the biological nano-magnetic particles are produced by 25-30mg per 1g of thallus, and the produced biological nano-magnetic particles can be used for in-vivo and in-vitro diagnostic reagents, drug carriers and the like.
The GKRZ vibrio with high yield of biological nano-magnetic particles is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No.19304,
the vibrio GKRZ of the high-yield biological nano-magnetic particles of the embodiment is separated and obtained from the water sample of the Kyoto aqueduct.
Inoculating the strain into a liquid culture medium with the inoculation amount of 5-10% (v/v), carrying out shake culture at the temperature of 28 ℃ and the rpm of 120 for 18 hours to logarithmic phase, and screening to obtain the Vibrio GKRZ with high-yield biological nano-magnetic particles by using a liquid magnetophoresis separation method, wherein the liquid culture medium comprises the following components: per liter, 3g of sodium pyruvate, 0.1g of KH2PO4, 0.15g of MgSO4.7H2O, 0.34g of NaNO3, 2.38g of hydroxyethyl piperazine ethanesulfonic acid, 3g of bean cake powder and 0.1g of yeast powder, and the pH value is adjusted to 6.0-7.0; the incubation temperature was 28 ℃.
Example 2 high yield biological nano-magnetic particle GKRZ Vibrio liquid magnetophoretic separation
Filtering the bacteria solution with the logarithmic growth period cultured in the embodiment 1 by using a microporous filter membrane (the aperture is 0.22 mu m) to obtain the Vibrio GKRZ with high yield of biological nano-magnetic particles;
the invention finally obtains the GKRZ vibrio with high yield of biological nano-magnetic particles by 67 generations of screening through a liquid magnetophoresis separation method.
The Vibrio GKRZ with high yield of biological nano-magnetic particles is preserved in China general microbiological culture Collection center (CGMCC) in 1 month and 3 months in 2020 at the address of: the preservation number of the institute of microbiology of the Chinese academy of sciences is CGMCC No. 19304.
The GKRZ vibrio with high yield of biological nano-magnetic particles has the following characteristics by identification:
gram staining is negative, anaerobic, acidophilic, rod-shaped, the size is (0.4-0.6 μm) x (1-3 μm), can use silicate as main energy substance, can grow in liquid culture medium and solid culture medium, the bacterial liquid is black, the bacterial colony is milky smooth bacterial colony, the optimum growth temperature is 28 ℃, and the optimum growth pH value is 6.5.
Example 3
The embodiment provides a method for extracting biological nano magnetic particles, which comprises the following steps:
(1) culturing Vibrio GKRZ by the method of example 1;
(2) centrifuging at 5000rpm for 10min, and collecting thallus;
(3) adding PBS buffer solution to homogenize and crush the thalli, wherein the mass ratio of the adding amount of the buffer solution to the GKRZ vibrio is 10:1, and collecting biological nano magnetic particles;
(4) adding PBS buffer solution for ultrasonic cleaning under the condition of 40KHz for 30min, and performing magnetic separation to obtain purified biological nano-magnetic particles.
Example 4
The embodiment provides a method for coupling biological nano-magnetic particles with a drug, wherein the drug is tocuzumab, and the method comprises the following steps: taking 1mg of the biomagnetic nanoparticles prepared in the example 1 and 1mg of SPDP for ultrasonic coupling, performing ultrasonic coupling for 1min each time, repeating the ultrasonic coupling for 30 times, and washing the biomagnetic nanoparticles for three times by using PBS (phosphate buffer solution) with the pH value of 7.4; adding 1mg of recombinant SPA for ultrasonic coupling, performing ultrasonic coupling for 1min each time, repeating for 30 times, and washing with PBS (phosphate buffer solution) with pH of 7.4 for three times; and then adding 1mg of trastuzumab for coupling, and placing the mixture in a shaking table at 37 ℃ for mixing for 2h to obtain the trastuzumab particle.
When the method of the embodiment is used for coupling the tuzumab, the encapsulation efficiency reaches 90 percent, is improved by 30 percent compared with the standard encapsulation efficiency, the drug loading rate reaches 90 percent, and is improved by 50 percent compared with the standard drug loading rate.
Example 5
In this example, the GKRZ vibrio cultured to the logarithmic growth period in example 1 was collected, the content of the biological nano-magnetic particles in the bacteria was observed, and the content of the biological nano-magnetic particles in the ordinary GKRZ vibrio was also observed, as a result, it was found that 30 to 50 biological nano-magnetic particles were contained in the GKRZ vibrio of example 1, and 10 to 25 biological nano-magnetic particles were contained in the ordinary GKRZ vibrio, and thus it could be proved that the yield of the biological nano-magnetic particles of the GKRZ vibrio provided by the present invention was significantly improved compared to the ordinary GKRZ vibrio.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (7)
1. The GKRZ vibrio with high yield of biological nano-magnetic particles is characterized in that the preservation number of the GKRZ vibrio with high yield of biological nano-magnetic particles is CGMCC No. 19304.
2. The high yield biomagnetic nanoparticle-producing vibrio GKRZ of the claim 1, wherein the gram stain is negative, anaerobic, acidophilic, rod-shaped, has the size of (0.4-0.6 μm) x (1-3 μm), can use silicate as a main energy substance, can grow in both liquid and solid media, and has black bacteria liquid and milky-white and smooth colonies.
3. The use of Vibrio GKRZ highly productive biological nano-magnetic particles as claimed in claim 1 or 2 in the production of recombinant biological nano-magnetic particle crystals.
4. The method for cultivating high yield biological nano magnetic particle vibrio GKRZ as claimed in claim 1 or 2, wherein the cultivation method is inoculating the vibrio GKRZ in liquid culture medium for fermentation cultivation.
5. A method for producing bio-nanomagnetic particles, the method comprising the steps of: (1) culturing the vibrio GKRZ of claim 1 or 2; (2) centrifugally collecting thalli; (3) adding buffer solution to homogenize and crush thallus and collecting nanometer magnetic particle; (4) adding buffer solution for ultrasonic cleaning, and performing magnetic separation to obtain purified biological nano magnetic particles.
6. A method for preparing a biological nano magnetic particle drug carrier is characterized by comprising the following steps: s1 culturing the Vibrio GKRZ of claim 1 or 2; s2 preparing biological nano magnetic particles by using GKRZ vibrio; s3 coupling the biological nano magnetic particle with the medicine.
7. The method for preparing biomagnetic particle drug-loaded according to claim 6, wherein the drug is tocuzumab, and the step S3 comprises the following specific steps: ultrasonically coupling biological nano magnetic particles with the SPDP in a mass ratio of 1:1 for 1min every time, repeating for 30 times, and washing for three times by using PBS with the pH value of 7.4; adding recombinant SPA with the same amount as the biological nano-magnetic particles and the SPDP, performing ultrasonic coupling for 1min each time, repeating for 30 times, and washing with PBS with pH of 7.4 for three times; and then adding trastuzumab with the same quantity as the biological nano magnetic particles and the SPDP for coupling, and placing in a shaking table at 37 ℃ for mixing for 2h to obtain the magnetic nanoparticle.
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